Regulation of inflammatory genes in decidual cells: Involvement of the bromodomain and extra-terminal family proteins

The decidua undergoes proinflammatory activation in late pregnancy, promoting labor. Bromodomain and Extra-Terminal (BET) family proteins interact with acetylated histones and may control gene expression in inflammation. Here, we assessed whether BETs are involved in inflammatory gene regulation in human decidual cells. We have treated primary cultures of decidual stromal cells (DSCs) from term pregnancies with endotoxin (LPS) and measured the expression of a panel of pro-and anti-inflammatory genes. BET involvement was assessed using the selective BET inhibitors (+)-JQ1 and I-BET-762 or the negative control compound (-)-JQ1. Histone 3 and -4 acetylation and BETs binding at the target gene promoters were determined to assess whether these processes are involved in the actions of LPS, BETs, and BET inhibitors. LPS increased the expression of the proinflammatory (PTGS2, IL6, CXCL8/IL8, TNF) and the anti-inflammatory (IL10, IDO1) genes of the panel. The constitutively expressed inflammatory genes (PTGS1, PTGES) were unaffected. The BET inhibitors, but not the control compound, reduced the basal and LPS-induced expression of PTGS1, PTGS2, IL6, CXCL8/IL8, IL10, and IDO1. TNF expression was not changed by BET inhibition. The dominant BETs were Bromodomain-containing protein -2 (BRD2) and -4L (BRD4L) in DSCs. LPS increased histone 4 acetylation at the CXCL8/IL8 and TNF promoters and histone 3 and -4 acetylation at the IDO1 promoter, while (+)-JQ1 abrogated histone acetylation at several promoters. Overall, histone acetylation and promoter binding of BETs showed no consistent relationship with gene expression across the gene panel and the treatments. BET proteins, predominantly BRD2 and BRD4L, control critical pro- and anti-inflammatory genes in DSCs. TNF induction exemplifies a BET-independent pathway. Changing histone acetylation at the promoters is not a general obligatory requirement for inflammatory gene expression in response to LPS. BETs likely act at chromatin loci separate from the examined promoters. BET inhibitors may block decidual activation at labor.


Introduction
The decidua is the endometrium of pregnancy, forming the maternal side of the maternal-fetal interface. It plays a crucial role in protecting the semi-allogenic fetus from immune rejection while preserving decidual competence to elicit an immune response against infections ascending from the vagina or originating from peripheral sources. These contrasting roles are performed by tightly controlled pro-and anti-inflammatory, immunosuppressive and immunepromoting responses [1,2]. Disturbances of this balance are associated with pregnancy disorders such as miscarriage, pre-eclampsia, early membrane rupture, and premature labor.
The decidua is a complex tissue with diverse and dynamic cellular composition. It contains a variety of bone marrow-derived CD45 + leukocytes that include monocytes/macrophages, dendritic cells, granulocytes, NK (natural killer) cells, T and B lymphocytes, and mast cells that change in abundance during gestation [3]. The largest proportion of cells is a resident decidual cell type establishing the stroma of the tissue. Decidual stromal cells (DSCs) exhibit pro-and anti-inflammatory capacities and influence leukocytes locally to adapt to the idiosyncratic needs of pregnancy [1]. DSCs promote Th2 immune bias [4], inhibit natural killer (NK) cell cytotoxicity, and reduce the activity of antigen-presenting (dendritic) cells [5] at the maternalfetal interface favoring pregnancy maintenance. The pregnancy-protective phenotype of the decidua vanes towards the end of gestation [6,7], and decidual cells shift to a proinflammatory phenotype [8], which promotes myometrial activation and cervical remodeling. This change is part of the process leading to labor and birth. Pathological activation of the decidua leads to dysfunctional labor, untimely delivery, and newborn complications. The mechanisms that underlie the inflammatory transformation of the decidua in normal or pathological labor are poorly understood. In culture, DSCs respond to LPS (lipopolysaccharide, an inflammatory trigger from Gram-negative bacteria) or cytokines like IL-1 beta and TNF-a by producing a variety of proinflammatory factors, including IL-6, IL-8, PTGS2, PTGES [9][10][11][12][13]. Anti-inflammatory cytokine production (IL-10) has also been reported after LPS treatment [14], indicating a balanced pro-and anti-inflammatory response.
Inflammatory transformation of cells involves widespread gene expression changes associated with alterations of chromatin structure at promoters, enhancers, and super-enhancers controlling inflammatory genes [15,16]. The altered acetylation state of histones and non-histone proteins has been recognized as a critical component of the process [17]. One initial event is the binding of BRD4, (bromodomain-containing protein 4) a member of the BET (bromodomain and extra-terminal) family of proteins, to gene regulatory regions in a complex with NFkB (nuclear factor NF-kappa-B), the central regulator of inflammation [18]. BET recruitment is critical for activating super-enhancers and promoters controlling proinflammatory genes [19,20], while BETs dissociating from other super-enhancers deactivates non-inflammatory genes during the phenotype transition [21]. BET family proteins bind acetyl-lysine residues in acetylated histone and non-histone proteins through their tandem bromodomains and protein interaction domains [22], which is the mechanistic basis of their chromatin interactions. Importantly, BET proteins can be targeted effectively by drugs that disrupt the bromodomain-acetyl lysine binding [23,24], which abrogates the super-enhancer mediated cell phenotype transitions [19]. BET proteins are emerging central players in inflammation with modifiable activity [25].
The role of BET proteins in inflammatory gene regulation in the decidua is undefined. Here we have addressed this gap of knowledge by determining (1) the effect of BET inhibitors on LPS-induced inflammatory gene expression in primary cultures of DSCs, (2) the expression of BET family members in DSCs, and (3) the effects of LPS and BET inhibitor on histone acetylation and BET protein binding at the promoters of inflammatory genes in the DSCs. The exclusion). The absence of CD45 + cells was verified by flow cytometry in preliminary experiments. A representative example of the flow cytometry analysis of freshly isolated decidual cells is presented in S1 Fig, showing that >99% of live single cells were CD45 negative after purification.
Cell culture. Cells were seeded at 10 5 /cm 2 in cell culture flasks with gelatin (Type-2, Sigma G1393)-coated culture surface. Cultures were incubated in a humidified atmosphere of 5% CO 2 in air at 37˚C. The medium was changed every third day until 10-14 days of culture. Immunocytochemical characterization of the sub-confluent cultures (S2 Fig) showed pervasive expression of vimentin, a marker of stromal cells in the decidua [26], and the absence of CD45 + cell contamination. The cultured DSCs were treated with (+)-JQ1, I-BET-762, or (-)-JQ1 (0.5 μM) for 48 h, followed by stimulation with lipopolysaccharide (LPS, from E. coli 055:B5, 1 μg/mL, for 24 h in the presence of the drugs).

Messenger RNA determination
Total RNA was isolated from cells cultured in T25 flasks using the RNeasy Mini Kit and the RNase-Free DNase Set (Qiagen, Chadstone Centre, VIC Australia) following the manufacturer's protocols. RNA purity was assessed by UV absorption using a NanoDrop 1000 spectrophotometer, and the Agilent 2100 Bioanalyzer System was used to verify RNA integrity. Purified RNA was spiked with Alien RNA transcript (10 7 copies/μg, Stratagene, Integrated Sciences, Chatswood NSW Australia), which served as the reference RNA in the qRT-PCR assays.
The RNA was reverse transcribed using the Superscript™ III (Life Technologies, Mulgrave, VIC Australia) with random hexamer primers. Quantitative real-time PCR was performed using the Applied Biosystems™ QuantStudio™ 6-Flex Real-Time PCR system (Thermo Fisher, Scoresby VIC Australia) with SYBR™ Green detection reagents sourced from the manufacturer. Primers have been designed using Primer-BLAST from NCBI, and their sequences are listed in the S1 Table. Primer and template cDNA concentrations were optimized for each mRNA assay [27].

Chromatin immunoprecipitation (ChIP)
Cell fixation. Cells grown in T75 culture flasks were fixed by a two-step cross-linking procedure that enhances the detection of modified histones and transcription factors bound to the chromatin [28]. Briefly, the adherent cultures were rinsed with PBS (phosphate-buffered saline) and cross-linked by adding 2 mM EGS (Ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester) (Sigma-Aldrich E3257) in PBS for 30 min at room temperature. Next, fresh buffer with 1% formaldehyde was added at room temperature for 10 min to perform the second cross-linking step. Formaldehyde action was quenched with 125 mM glycine in cold PBS. Cells were permeabilized in hypotonic buffer (50 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl 2 , 0.15% v/v Igepal Ca-630) supplemented with protease inhibitors (cOmplete™ Mini Protease Inhibitor Cocktail, Roche, and 0.5 mM phenylmethanesulfonyl fluoride, PMS) and histone deacetylase inhibitor (10 mM Na-butyrate). Cells were scraped off the culture surface, and the nuclei were sedimented by centrifugation and stored at -80˚C until further processing.
Preparation of chromatin extracts. Nuclear pellets were suspended in Extraction Buffer (50 mM Tris HCl, pH8.1; 10 mM EDTA; 1% SDS and inhibitors as above) and sonicated using a Misonix XL-2000 microprobe sonifier with the following optimized settings: Ten 10-sec bursts at 8-11 watts and one pulse/sec, separated by 50-sec intervals. Heating was prevented by performing the sonication in a salt-ice bath. Sonicated samples were centrifuged at 13,000 rpm at 4˚C for 30 min, and the supernatants were stored frozen until immunoprecipitation.
Chromatin elution, crosslink reversal, and DNA purification. Chromatin fragments were eluted by incubating the beads in 1% SDS, 0.1 M NaHCO 3 at 65˚C for 10 min. The beadfree eluates were supplemented with Proteinase K (57 μg/mL) and incubated at 56˚C for one hour, followed by 65˚C overnight to reverse crosslinks. An aliquot of each sonicated extract was processed for crosslink reversal without immunoprecipitation to determine the input in the ChIP assays.
DNA was purified by phenol-chloroform-isoamyl alcohol extraction (25:24:1 v/v) followed by Na-acetate-ethanol precipitation with 45 μg GlycoBlue™ (Invitrogen) co-precipitant added to each sample. Precipitates were washed 2 x with TE buffer to remove residual SDS and dissolved in TE. DNA content in the input samples was determined by UV absorption using the NanoDrop 1000 spectrophotometer.
Quantitative real-time PCR (qPCR). The abundance of target gene promoter sequences in the immunoprecipitated DNA was determined by qPCR. Gene-specific oligonucleotide primer sequences and their positions relative to transcription start sites are listed in S1 Table. Oligonucleotide primers were designed and optimized as described above. Triplicate PCR reactions were performed with each chromatin extract and gene from each immunoprecipitation using the QuantStudio™ 6-Flex Real-Time PCR system with SYBR™ Green detection.

Data processing and statistical analysis
Messenger RNA abundance. Results were calculated relative to the spiked-in exogenous RNA [30] (Alien) by the delta-Ct method [27]. Biological variation was reduced by scaling data over the average of all treatments in each experiment. Data were tested for distribution and transformed to meet the criteria for normal distribution. The data were then analyzed by repeated-measures ANOVA followed by F-tests for individual contrasts with significance adjusted to false discovery rate (FDR, "Q") using the Benjamini-Hochberg method. In some of the experiments, as indicated in the Figure legends, the non-normally distributed data were evaluated using the non-parametric K-sample-equality-of-medians test followed by Fisher's exact test, Wilcoxon's signed-rank test, or Dunn's test of multiple comparisons using rank sums with Bonferroni-adjustment of significance, as appropriate. Differences at p<0.05 or Q<0.05 were considered statistically significant.
ChIP. Results were calculated as the recovery of promoter fragments relative to input using delta-Ct values. The results for each gene were scaled over the average of all treatments with each immunoprecipitation (acetyl-histone 3, acetyl-histone 4, BRD2, BRD4, IgG) in each experiment and expressed relative to the negative control, IgG, to reduce biological and technical variation. Data were tested for distribution and transformed to meet normal distribution criteria. The data were then analyzed by repeated-measures ANOVA followed by F-tests for individual contrasts with significance adjusted to false discovery rate (FDR, "Q") using the Benjamini-Hochberg method. Q<0.05 (5% FDR) was considered statistically significant. Statistical calculations were performed using STATA/IC 15.1 (College Station, TX; RRID: SCR_012763)

BETs are involved in gene expression control in DSCs
Treatments and gene targets. We have utilized two highly selective BET inhibitors, (+)-JQ1 and I-BET-762 [31,32], which can serve as chemical probes of functional BET involvement in chromatin regulation and gene expression control [33]. We have also treated cells with (-)-JQ1, the enantiomer of (+)-JQ1, as the inactive negative control compound [31]. Lipopolysaccharide (LPS) was used to model inflammatory stimulation by bacteria. We determined the effects of the treatments on a panel of pro-and anti-inflammatory genes selected for their potential participation in the inflammatory response of DSCs (S3 Fig). Basal levels of these mRNAs varied significantly in vehicle-treated cells (p = 0.001, K-sample-equality-ofmedians test), with the constitutively expressed PTGS1 and PTGES exhibiting the highest mRNA abundance. At the same time, the expression of the inducible PTGS2, TNF, and IDO1 genes was low.
The effects of LPS and BET inhibitors on target gene expression.

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inhibitors (+)-JQ1 and I-BET-762, but not the negative control (-)-JQ1, reduced the basal (drugs vs. vehicle) and LPS-stimulated (LPS + drugs vs. LPS) expression of PTGS2, IL6, and IL10. The drugs did not affect the low basal levels of CXCL8/IL8 and IDO1 mRNAs. Still, the LPS-induced expression of these genes was significantly blocked by the BET inhibitors but not the control compound. BET inhibitors did not affect the expression of TNF and PTGES either in the presence or absence of LPS. Finally, the abundance of PTGS1 mRNA was decreased by (+)-JQ1 both in the presence and absence of LPS, while (-)-JQ1 and I-BET-762 had no effect  under the treatment conditions employed. The response patterns of PTGS2, IL6, IL10, CXCL8/ IL8, and IDO1 to BET inhibitors strongly suggest that BET proteins are involved in the mechanism of LPS stimulation of these genes in the DSCs. The lack of effect of BET inhibitors indicates that TNF and PTGES expression is not BET dependent. Our data suggest some BET involvement in the (constitutive) expression of PTGS1.

BETs are expressed in DSCs
(+)-JQ1 and I-BET-762 are pharmacological probes targeting the tandem bromodomains of the BET family members BRD2, BRD3, BRD4, and BRDT (bromodomain-containing proteins 2, -3, -4, and bromodomain testis-specific protein) [33,34]. We have determined the expression of these BETs (except for the testis-specific BRDT) in DSCs to delineate the BET inhibitor targets that mediate the effects of the drugs. Data in Fig 5A) shows that BRD2, BRD3, and BRD4 mRNAs are present in DSCs. Both the long (canonical) BRD4 mRNA variant, BRDL, and the truncated BRD4S were detected. Differences in the mRNA levels indicated that BRD2 and BRD4L were the dominant BETs in the DSCs. LPS did not affect BRD2 and BRD4L expression (Fig 5B and 5C). Treatment with (+)-JQ1, but not with (-)-JQ1, significantly increased the level of BRD2 mRNA (Fig 5B), indicating BET-dependent negative feedback. BRD4L mRNA levels were not affected by the drugs (Fig 5C).

Histone acetylation and BET presence at target gene promoters
BET proteins bind acetylated histone 3 and -4 (acH3 and acH4) through their bromodomains, which is critical to their actions in chromatin. BRD2 and -4 have been reported to bind and interact with the promoters of genes they control [35,36]. Based on these observations, we have explored histone acetylation and BRD2 and -4 presence at the promoters of the genes induced by LPS in our gene panel. Chromatin immunoprecipitation (ChIP) was performed using antibodies that recognize acH3, acH4, BRD2, and BRD4, and the enrichment of proximal promoter sequences of the target genes relative to the negative control IgG was deter- We have detected histone 3 acetylation at the IL6, CXCL8/IL8, and TNF promoters in unstimulated DSCs (vehicle vs. the ChIP negative control, IgG). Histone 3 acetylation was not detected at the promoters of the anti-inflammatory IL10 and IDO1 genes in unstimulated cells. LPS treatment induced histone 3 acetylation at the IDO1 promoter but not at the other promoters (LPS vs. Vehicle). Treatment with (+)-JQ1 alone resulted in a significant decrease in acH3 level at the IL6 promoter, which was also seen in the presence of the negative control compound, (-)-JQ1 ((+)-JQ1 or (-)-JQ1 vs. Vehicle). Conversely, (+)-JQ1 alone increased histone 3 acetylation at the IL10 and IDO1 promoters, but this effect was not shared by (-)-JQ1. Treatment with (-)-JQ1, but not with (+)-JQ1, reduced acH3 levels at the IL6 and CXCL8/IL8 promoters after LPS exposure (LPS + (+)-JQ1 or (-)JQ1 vs. LPS). Furthermore, (+)-JQ1 increased histone 3 acetylation at the TNF and IL10 promoters in response to LPS, but the negative control drug (-)-JQ1 was ineffective at these sites. At the IDO1 promoter, histone 3 acetylation after LPS treatment was unaffected by the drugs. Overall, the differences in Histone 3 In unstimulated cells, we have detected histone 4 acetylation at the IL6 promoter (relative to IgG). After LPS stimulation, the acH4 levels increased significantly at the TNF and the IDO1 promoters relative to the vehicle. Notably, the acH4 level at the CXCL8/IL8 promoter was significant in LPS-treated cells relative to the IgG negative control but not compared to vehicle-treated cells, which had an acH4 level similar to IgG, suggesting indirectly that LPS stimulated histone 4 acetylation at this locus, as well. Treatment with (+)-JQ1 alone increased histone 4 acetylation of the IL10 and the IDO1 promoters relative to the vehicle, while (-)-JQ1 did not show this effect. At the IL6 promoter, (-)-JQ1, but not its active analog, (+)-JQ1, reduced histone 4 acetylation relative to vehicle treatment ( Fig 10A). The drugs did not influence histone 4 acetylation at the CXCL8/IL8, IL10, and IDO1 promoters in response to LPS. At the IL6 promoter, treatment with (-)-JQ1, but not with (+)-JQ1, reduced acH4 levels in response to LPS. Like with histone 3, histone 4 acetylation changes at the promoters showed no consistent relationship to the gene expression changes after the same treatments.
BRD2 and BRD4. ChIP analysis with antibodies verified for chromatin immunoprecipitation did not detect significant BRD2 or BRD4 binding to the promoter of the investigated genes at the 5% FDR level under our experimental conditions. The contrast lists of BRD2 and BRD4 ChIP results are provided in S7 and S10 Figs, respectively. The diagrams showing the cells (Dunn's test of multiple comparisons using rank sums with Bonferroni adjustment of significance). The results of the individual experiments and summary statistics are presented in the S1 Data.
https://doi.org/10.1371/journal.pone.0280645.g005   Table), and the results were expressed relative to the negative control IgG. Further details of the experimental procedures and the data processing are described in the Materials and methods section. Columns show the mean ± SEM, n = 5-6 independent experiments. Significant differences (at 5% FDR), highlighted in the heatmap in Fig 6, are indicated by the horizontal orange and blue lines. The results of the individual experiments and summary statistics are presented in the S1 Data. BRD2 and BRD4 ChIP results in individual gene promoters under all treatment conditions are presented in S8 and S9 Figs for BRD2 and S11 and S12 Figs for BRD4.

Discussion
Pharmacological agents that bind the tandem bromodomains of the BET family transcription factors have been used successfully to determine the involvement of these proteins in regulatory events at the molecular level. Blocking the bromodomains interferes with the binding of BETs to lysine-acetylated histones disrupting their gene regulatory function. Two of the best-

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characterized members of the BET inhibitors are (+)-JQ1 and I-BET-762. When these compounds are employed at a concentration of � 1 μM, and their effects are contrasted to an analog of a similar structure practically inactive on the target, conclusions about functional BET involvement can be drawn with confidence. The verified control compound we used was (-)-JQ1, the enantiomer of (+)-JQ1 [31,37]. The pharmacological selectivity of BET inhibitors provided an opportunity to assess the involvement of the BET proteins in the regulation of inflammatory genes in DSCs critical for pregnancy maintenance and the timing of labor [7,38,39].
We have established primary cultures of decidual stromal cells isolated from term placentas by adapting published procedures [10]. Cells were purified by removing CD45 + leukocytes and cultured without passage in the presence of estrogen and progestogen for a maximum of 20 days to approach conditions preserving the in vivo DSC phenotype. LPS was employed as the inflammatory stimulus to model infectious insults the decidua may be exposed to under pathological conditions. We chose to determine treatment effects on genes prototypical of pro-and anti-inflammatory responses. The target genes included PTGS1, PTGS2, and PTGES, coding for Prostaglandin G/H Synthase 1, -2; and Prostaglandin E Synthase, which are critical enzymes of the prostaglandin synthesis pathway; IL6 (encoding Interleukin-6, a multifunctional proinflammatory cytokine), CXCL8/IL8 (responsible for producing Interleukin-8, a neutrophil chemokine and proinflammatory factor), and TNF (the gene for Tumor Necrosis Factor, a pleiotropic cytokine critically involved in inflammatory responses). We have also studied genes encoding anti-inflammatory factors such as IL-10 (Interleukin-10) and IDO-1 (Indoleamine 2,3-Dioxygenase 1), a secreted tryptophan metabolizing enzyme with potent immune-mitigating and antimicrobial actions [40].
LPS stimulated the expression of all studied genes except PTGS1 and PTGES, which had constitutively high mRNA levels. Notably, both the pro-inflammatory genes (PTGS2, IL6, CXCL8/IL8, TNF) and the anti-inflammatory genes (IL10 and IDO1) in our panel responded to LPS treatment. The responses were robust, even considering that the primary cells could   Table), and the results were expressed relative to the negative control IgG. Further details of the experimental procedures and the data processing are described in the legend of Fig 7. Columns show the mean ± SEM, n = 5-6 independent experiments. Significant differences (at 5% FDR), highlighted in the heatmap in Fig  9, are indicated by the horizontal orange and blue lines. The results of the individual experiments and summary statistics are presented in the S1 Data.
https://doi.org/10.1371/journal.pone.0280645.g010 have been exposed to infectious stimuli in situ before isolation. We took care to process tissues after elective operative (Cesarean) deliveries without clinical or histological signs of intrauterine infection or inflammation to minimize the risk of in situ LPS exposure. Nevertheless, this possibility represents a limitation of the study. The BET inhibitor effects on mRNA levels were consistent with the involvement of BETs in the LPS responses except for TNF, which was not influenced significantly by (+)-JQ1, I-BET-762, or (-)-JQ1. PTGS2, IL6, and IL10 mRNA levels were reduced by (+)-JQ1 and I-BET-734, but not (-)-JQ1, in unstimulated cells, indicating BET involvement in basal expression. The lack of BET inhibitor effect on TNF expression suggests that there is a BET-independent pathway of LPS action in DSCs. TNF-a has been reported to stimulate the IL-6 [41], IL-8 [9], IL-10 [42], and prostaglandin [42][43][44][45] production of decidual cells or tissue in vitro, and time course and immunoneutralization experiments suggested that TNF-a may mediate or augment the action of LPS on inflammatory mediator release from choriodecidua explants [42]. Moreover, TNF-a has been shown to promote the proinflammatory transformation of endothelial (HUVEC) cells by a mechanism that includes the redistribution of BRD4, one of the BETs dominantly expressed in decidual cells, to superenhancer chromatin loci controlling inflammatory genes [19]. Our data are compatible with an LPS-induced, TNF-a-mediated, and BRD4 -dependent mechanism of inflammatory transition of DSCs. Firm evidence for this scenario will require integrated genome-wide data of transcriptome changes, chromatin remodeling, and BET localization in response to LPS and other labor-associated stimulants.
The chromatin immunoprecipitation study aimed to gain molecular-level information about BET involvement in LPS actions on our inflammatory gene panel. BET involvement in gene regulation is diverse and multifaceted [22]. We have focused on the proximal promoter regions (within 1kb upstream of the transcription start site) where BRD2 and BRD4 were reported to control many genes [36,46]. Nonetheless, we detected no significant binding of BRD2 or BRD4 at 5% FDR to the proximal promoter of the investigated genes. However, a low level of binding cannot be excluded because some unadjusted significance values were below the P = 0.05 cut-off (S5 and S7 Figs). The BRD2 and the BRD4 antibodies have been ChIP certified, and we have employed a dual fixation procedure to detect both direct and indirect (protein-to-protein mediated) binding to DNA [47]. We cannot exclude that technical reasons, e.g., epitope masking, may have contributed to the low ChIP signal. Furthermore, we have assayed BET binding after extended treatments optimized for mRNA changes, which could not detect possible early transient BRD binding event(s). Establishing the time course of BET binding to a broader array of candidate genes and regulatory chromatin regions after stimulation, especially enhancers and super-enhancers [21,22], is a critical next approach to delineating the relationship between chromatin-bound BRDs and target gene expression in DSCs.
Histone 3 acetylation was detected at the promoters of three of the five genes examined (IL6, CXCL8/IL8, TNF). LPS did not affect acH3 levels at these promoters or at the promoter of IL10, which was not acetylated in the unstimulated state. The expression of these genes, however, was significantly enhanced by LPS. In our limited panel of LPS target genes, only IDO1 exhibited a concomitant increase in mRNA expression and promoter histone 3 acetylation after endotoxin treatment. This indicates that histone 3 acetylation at the promoter is not a general obligatory requirement for LPS-induced inflammatory gene expression in DSCs. A similar conclusion can be drawn about histone 4 acetylation, which was found only at the IL6 promoter in the unstimulated state and was induced by LPS only at the TNF and CXCL8/IL8 promoters (vs. the IgG control) despite upregulated expression across the panel of genes analyzed by ChIP.
BET inhibitor treatment abrogated histone acetylation at the inflammatory gene panel. One type of response was observed at the IL10 and IDO1 promoters, where (+)-JQ1 increased H3 and H4 acetylation in unstimulated cells. Treatment with (+)-JQ1 enhanced H3 acetylation at the IL10 and TNF promoters in response to LPS. (-)-JQ1 did not show these effects, arguing for BET involvement, which was likely indirect since no BRD binding was detected at these sites. BRD4 has been reported to possess an intrinsic histone acetyltransferase activity [48] and augment histone acetylation by interacting with p300 and CBP [49], but the mechanisms involved in DSCs remain to be established. Notably, acetyl-H3 and -H4 levels at the promoters showed no consistent relationship with gene expression changes. The second type of response was detected at the IL6, and CXCL8/IL8 promoters, where the negative control compound, (-)-JQ1, but not its active analog, (+)-JQ1, reduced histone acetylation in LPS stimulated cells.
Furthermore, both (+)-JQ1 and (-)-JQ1 decreased IL6 promoter acH3 levels in unstimulated cells. These actions of (-)-JQ1, even when copied by (+)-JQ1, may be off-target, not involving BETs. Alternatively, molecular dynamics (MD) simulations assessing the binding energies of (+)-JQ1 and (-)-JQ1 to the BRD4 bromodomain have indicated that both compounds possess similar affinities to BRD4 [50]. The more than 100-fold higher IC 50 of (-)-JQ1 compared to its chiral isomer, (+)-JQ1 [31], could be attributed to the faster insertion of (+)-JQ1 into the binding pocket of the bromodomain [50]. Despite the slower binding kinetics (-)-JQ1 might still be able to associate with BRDs after more prolonged exposure, such as 72 h in our experiments (48 h pre-treatment and 24 h co-treatment with LPS), especially since the bromodomain dissociation kinetics of (-)-JQ1 is also slower than that of (+)-JQ1 [50]. The MD simulation results may thus allow BET-mediated actions by (-)-JQ1, depending on experimental conditions. Again, the drug-induced changes in acetyl histone levels at the promoters did not correlate with gene activity.
In summary, our results demonstrate that BET proteins are critically involved in the response of DSCs to inflammatory stimulation. Using a panel of candidate genes, we show that both pro-and anti-inflammatory genes respond in a BET-dependent fashion and BET inhibitor drugs inhibit their responses. A BET-independent pathway also exists, mediating the stimulation of TNF expression by LPS. Analysis of acetylated histone levels at the promoters indicates that enhanced histone acetylation is not involved obligatorily in the transcriptional responses. BETs may regulate gene activity by acting at distant (e.g., enhancer) loci rather than at the proximal promoters. Nevertheless, BET inhibitors may represent a new class of pharmacological agents capable of arresting the inflammatory transformation of the decidua at labor with potential therapeutic benefit. Purified decidual stromal cells were cultured to near confluence as described in the Materials and methods section. Cultures were fixed with paraformaldehyde, permeabilized, and blocked with hydrogen peroxide, followed by antigen retrieval using standard procedures (Citric acid, pH 6.0, 95C for 15 min). Non-specific staining was blocked with 10% goat serum. Immunocytochemistry was performed using the VECTASTAIN ABC kit (PK6102, Vector Laboratories) following the manu-  stromal cells from term decidua were cultured to near confluence, and  PTGS1, PTGS2, PTGES, IL6, CXCL8/IL8, TNF, ILI0, and IDO1 mRNA abundance was determined by qRT-PCR relative to a spiked-in reference RNA (Alien). Values were scaled relative to the average in each experiment. Columns represent the mean + SEM, n = 5 independent experiments. The abundance of the mRNAs is significantly different from each other (p = 0.001, K-sample-equality-of-medians test).  Table), and the results were expressed relative to the negative control IgG. Columns show the mean ± SEM, n = 5 independent experiments. No significant BRD2 binding relative to IgG was detected (at 5% FDR) under any treatment condition. The results of the individual experiments and summary statistics are presented in the S1 Data. Chromatin immunoprecipitation (ChIP) was performed using a ChIP-certified BRD2 antibody (S2 Table), and the results were expressed relative to the negative control IgG. Columns show the mean ± SEM, n = 4-5 independent experiments. No significant BRD2 binding relative to IgG was detected (at 5% FDR) under any treatment condition. The results of the individual experiments and summary statistics are presented in the S1 Data.  Table), and the results were expressed relative to the negative control IgG. Columns show the mean ± SEM, n = 5-6 independent experiments. No significant BRD4 binding relative to IgG was detected (at 5% FDR) under any treatment condition. The results of the individual experiments and summary statistics are presented in the S1 Data. Chromatin immunoprecipitation (ChIP) was performed using a ChIP-certified BRD4 antibody (S2 Table), and the results were expressed relative to the negative control IgG. Columns show the mean ± SEM, n = 5-6 independent experiments. No significant BRD4 binding relative to IgG was detected (at 5% FDR) under any treatment condition. The results of the individual experiments and summary statistics are presented in the S1 Data. (TIF) S1